Association of Na+-H+ Exchanger Isoform NHE3 and Dipeptidyl Peptidase IV in the Renal Proximal Tubule

In an attempt to identify proteins that assemble with the apical membrane Na+-H+ exchanger isoform NHE3, we generated monoclonal antibodies (mAbs) against affinity-purified NHE3 protein complexes isolated from solubilized renal microvillus membrane vesicles. Hybridomas were selected based on their ability to immunoprecipitate NHE3. We have characterized in detail one of the mAbs (1D11) that specifically co-precipitated NHE3 but not villin or NaPi-2. Western blot analyses of microvillus membranes and immunoelectron microscopy of kidney sections showed that mAb 1D11 recognizes a 110-kDa protein highly expressed on the apical membrane of proximal tubule cells. Immunoaffinity chromatography was used to isolate the antigen against which mAb 1D11 is directed. N-terminal sequencing of the purified protein identified it as dipeptidyl peptidase IV (DPPIV) (EC 3.4.14.15), which was confirmed by assays of DPPIV enzyme activity. We also evaluated the distribution of the NHE3-DPPIV complex in microdomains of rabbit renal brush border. In contrast to the previously described NHE3-megalin complex, which principally resides in a dense membrane population (coated pits) in which NHE3 is inactive, the NHE3-DPPIV complex was predominantly in the microvillar fraction in which NHE3 is active. Serial precipitation experiments confirmed that anti-megalin and anti-DPPIV antibodies co-precipitate different pools of NHE3. Taken together, these studies revealed an unexpected association of the brush border Na+-H+ exchanger NHE3 with dipeptidyl peptidase IV in the proximal tubule. These findings raise the possibility that association with DPPIV may affect NHE3 surface expression and/or activity. In an attempt to identify proteins that assemble with the apical membrane Na+-H+ exchanger isoform NHE3, we generated monoclonal antibodies (mAbs) against affinity-purified NHE3 protein complexes isolated from solubilized renal microvillus membrane vesicles. Hybridomas were selected based on their ability to immunoprecipitate NHE3. We have characterized in detail one of the mAbs (1D11) that specifically co-precipitated NHE3 but not villin or NaPi-2. Western blot analyses of microvillus membranes and immunoelectron microscopy of kidney sections showed that mAb 1D11 recognizes a 110-kDa protein highly expressed on the apical membrane of proximal tubule cells. Immunoaffinity chromatography was used to isolate the antigen against which mAb 1D11 is directed. N-terminal sequencing of the purified protein identified it as dipeptidyl peptidase IV (DPPIV) (EC 3.4.14.15), which was confirmed by assays of DPPIV enzyme activity. We also evaluated the distribution of the NHE3-DPPIV complex in microdomains of rabbit renal brush border. In contrast to the previously described NHE3-megalin complex, which principally resides in a dense membrane population (coated pits) in which NHE3 is inactive, the NHE3-DPPIV complex was predominantly in the microvillar fraction in which NHE3 is active. Serial precipitation experiments confirmed that anti-megalin and anti-DPPIV antibodies co-precipitate different pools of NHE3. Taken together, these studies revealed an unexpected association of the brush border Na+-H+ exchanger NHE3 with dipeptidyl peptidase IV in the proximal tubule. These findings raise the possibility that association with DPPIV may affect NHE3 surface expression and/or activity. Na+-H+ exchanger NHE regulatory factor monoclonal antibody(ies) dipeptidyl peptidase IV polyacrylamide gel electrophoresis polyvinylidene difluoride The majority of NaCl, NaHCO3 and water filtered by the kidney is reabsorbed in the proximal tubule. Na+-H+ exchange is the predominant mechanism for absorption of Na+ and secretion of H+across the apical membrane of proximal tubule cells (1Alpern R.J. Physiol. Rev. 1990; 70: 79-114Crossref PubMed Scopus (190) Google Scholar). Several lines of evidence indicate that NHE3 is the Na+-H+exchanger isoform responsible for most, if not all, apical membrane Na+-H+ exchange activity in this segment of the nephron (2Biemesderfer D. Pizzonia J. Abu-Alfa A. Exner M. Reilly R. Igarashi P. Aronson P.S. Am. J. Physiol. 1993; 265: F736-F742PubMed Google Scholar, 3Amemiya M. Loffing J. Lotscher M. Kaissling B. Alpern R.J. Moe O.W. Kidney Int. 1995; 48: 1206-1215Abstract Full Text PDF PubMed Scopus (355) Google Scholar, 4Wu M.S. Biemesderfer D. Giebisch G. Aronson P.S. J. Biol. Chem. 1996; 271: 32749-32752Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 5Schultheis P.J. Clarke L.L. Meneton P. Miller M.L. Soleimani M. Gawenis L.R. Riddle T.M. Duffy J.J. Doetschman T. Wang T. Giebisch G. Aronson P.S. Lorenz J.N. Shull G.E. Nat. Genet. 1998; 19: 282-285Crossref PubMed Scopus (710) Google Scholar, 6Wang T. Yang C.L. Abbiati T. Schultheis P.J. Shull G.E. Giebisch G. Aronson P.S. Am. J. Physiol. 1999; 277: F298-F302Crossref PubMed Google Scholar, 7Choi J.Y. Shah M. Lee M.G. Schultheis P.J. Shull G.E. Muallem S. Baum M. J. Clin. Invest. 2000; 105: 1141-1146Crossref PubMed Scopus (112) Google Scholar). This isoform thereby plays an important role in the maintenance of fluid and electrolyte balance, and its activity is regulated in response to a wide variety of acute and chronic physiologic stimuli (8Alpern R.J. Yamaji Y. Cano A. Horie S. Miller R.T. Moe O.W. Preisig P.A. J. Lab. Clin. Med. 1993; 122: 137-140PubMed Google Scholar, 9Paillard M. Exp. Nephrol. 1997; 5: 277-284PubMed Google Scholar, 10Moe O.W. J. Am. Soc. Nephrol. 1999; 10: 2412-2425Crossref PubMed Google Scholar, 11Weinman E.J. Minkoff C. Shenolikar S. Am. J. Physiol. Renal Physiol. 2000; 279: F393-F399Crossref PubMed Google Scholar). The polarized expression and regulation of a transporter such as NHE3 necessarily involves interactions with other proteins. Recent studies have indicated that NHE3 is capable of binding calmodulin (12Wakabayashi S. Ikeda T. Noel J. Schmitt B. Orlowski J. Pouyssegur J. Shigekawa M. J. Biol. Chem. 1995; 270: 26460-26465Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), the NHE1 regulatory factor (NHERF) (11Weinman E.J. Minkoff C. Shenolikar S. Am. J. Physiol. Renal Physiol. 2000; 279: F393-F399Crossref PubMed Google Scholar, 13Weinman E.J. Steplock D. Donowitz M. Shenolikar S. Biochemistry. 2000; 39: 6123-6129Crossref PubMed Scopus (128) Google Scholar) and its homologue, exchanger-3 kinase A regulatory protein (E3KARP) (14Yun C.H. Oh S. Zizak M. Steplock D. Tsao S. Tse C.M. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (403) Google Scholar), and the calcineurin B homologous protein (15Pang T. Su X. Wakabayashi S. Shigekawa M. J. Biol. Chem. 2001; 276: 17367-17372Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). These interactions have generally been characterized in nonepithelial cells transfected to overexpress NHE3. We have been investigating whether NHE3 exists in assemblies with other proteins in native kidney membranes. We previously reported that the sedimentation coefficient for NHE3 solubilized from renal membranes is greater than predicted for monomeric NHE3, indicating the presence of multimeric complexes (16Biemesderfer D. Nagy T. DeGray B. Aronson P.S. J. Biol. Chem. 1999; 274: 17518-17524Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). To identify proteins that are associated with NHE3 in native renal kidney membranes, we generated monoclonal antibodies (mAbs) against affinity-purified NHE3 protein complexes from solubilized microvillus membranes. The study of one of these antibodies, mAb 10A3, demonstrated that a significant pool of NHE3 exists in a complex with the scavenger receptor megalin in the kidney brush border (16Biemesderfer D. Nagy T. DeGray B. Aronson P.S. J. Biol. Chem. 1999; 274: 17518-17524Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). We subsequently demonstrated that NHE3 is expressed in different microdomains of the brush border and that the NHE3-megalin complex principally resides in a dense membrane compartment most likely representing coated pits (17Biemesderfer D. DeGray B. Aronson P.S. J. Biol. Chem. 2001; 276: 10161-10167Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). We now report the detailed characterization of a second mAb (1D11) that specifically co-precipitates NHE3 from solubilized microvillus membranes isolated from rabbit renal cortex. We demonstrate that the antigen for mAb 1D11 is dipeptidyl peptidase IV (DPPIV, also known as CD26) and thus that NHE3 resides in an oligomeric complex with DPPIV in the renal proximal tubule. We also show that the NHE3-DPPIV complex is primarily located in the microvillar microdomain of the kidney brush border. These findings raise the possibility that association with DPPIV may affect NHE3 surface expression and/or activity. In a previous publication, we described in detail the development and characterization of monoclonal antibodies to a restricted region of the C terminus of NHE3 (18Biemesderfer D. Rutherford P.A. Nagy T. Pizzonia J.H. Abu-Alfa A.K. Aronson P.S. Am. J. Physiol. 1997; 273: F289-F299Crossref PubMed Google Scholar). mAb 2B9 was raised to a fusion protein (fpNHE3–702-832) that reproduced the C-terminal 131 amino acids of rabbit NHE3. This antibody was used as purified IgG from hybridoma supernatants. For immunoblotting experiments, we also used a previously characterized anti-NHE3 polyclonal antibody raised in goats to fpNHE3–702-832 (17Biemesderfer D. DeGray B. Aronson P.S. J. Biol. Chem. 2001; 276: 10161-10167Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). In a previous paper, we described the development and characterization of mAb 10A3 to rabbit megalin (16Biemesderfer D. Nagy T. DeGray B. Aronson P.S. J. Biol. Chem. 1999; 274: 17518-17524Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). A mAb (mouse IgG) to villin was purchased from AMAC, Inc. (Westbrook, Maine). A polyclonal antibody raised to the renal brush border Na/Pi cotransporter, NaPi-2, was a gift from Dr. Heini Murer and Dr. Jurg Biber, Institute of Physiology, University of Zurich-Irchel, Zurich, Switzerland (19Levi M. Lotscher M. Sorribas V. Custer M. Arar M. Kaissling B. Murer H. Biber J. Am. J. Physiol. 1994; 267: F900-F908PubMed Google Scholar). Horseradish peroxidase-conjugated rabbit anti-goat (heavy and light chain-specific), goat anti-mouse (γ chain-specific) and goat anti-rabbit (heavy and light chain-specific) were purchased from Zymed Laboratories Inc. (San Francisco, CA). Fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG (heavy and light chain) was also purchased from Zymed Laboratories Inc. (San Francisco, CA). The procedure for development of monoclonal antibodies against affinity-purified NHE3 complexes as well as the enzyme-linked immunosorbent assay performed for screening the hybridoma supernatants have been previously described in detail (16Biemesderfer D. Nagy T. DeGray B. Aronson P.S. J. Biol. Chem. 1999; 274: 17518-17524Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). mAbs were purified from hybridoma supernatants by affinity chromatography using protein G-Sepharose 4B (Pharmacia Biotech, Inc. Piscataway, NJ) according to manufacturer's protocols. Purified monoclonal antibody to which was was in kidney from were and in the A and membrane were using a based on precipitation and as previously described P.S. J. Biol. PubMed Scopus Google Scholar). experiments were as described previously D. DeGray B. Aronson P.S. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). renal microvillus membranes were solubilized in and as described The were to for or using a NJ) or a of antibodies were to the and the were for complexes were using of protein G-Sepharose 4B The were in and for and were solubilized in and proteins were by using polyacrylamide according to PubMed Scopus Google Scholar). For proteins were to polyvinylidene difluoride from polyacrylamide for with a electrophoresis Francisco, and with in of membranes proteins were in and in for to binding of by in antibodies were in in from to The membranes were in and for with peroxidase-conjugated IgG from Zymed Laboratories Inc. antibody was with according to manufacturer's protocols. to membranes for were in an In experiments membranes were and with antibodies The procedure of the membranes in for were with and in and was and using the as described previously D. G. Aronson P.S. M.G. Am. J. Physiol. Google Scholar). sections of were using a chromatography was by mAb 1D11 to protein a rabbit anti-mouse IgG C. U. J. Biol. Chem. Full Text PDF PubMed Google Scholar). membrane solubilized in and in were with the for The was and the was with NaCl, with and with antigen was by the with for The was with This was and the were and to of the chromatography by and to a membrane as the was from the blot for N-terminal amino sequencing by the and M. membranes were solubilized and to chromatography as of the of were for Western blot and enzyme of the of the solubilized microvillus membranes with the as well as the from the were to for used for Western blot and enzyme assays were for of microvillus membranes. the of fraction of were used for and and were used for of the enzyme dipeptidyl peptidase IV M. H. T. T. H. Clin. Chem. PubMed Scopus Google Scholar) and G. Clin. Chem. PubMed Scopus Google Scholar) were by the of from the of and For the DPPIV of fraction from the were to and The was for and by the of of For the of fraction were to of in and for of was based on the were and on as described previously (17Biemesderfer D. DeGray B. Aronson P.S. J. Biol. Chem. 2001; 276: 10161-10167Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). were from the and which are in were from in the (17Biemesderfer D. DeGray B. Aronson P.S. J. Biol. Chem. 2001; 276: 10161-10167Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). In a previous paper, we described the of mAbs against affinity-purified NHE3 protein complexes (16Biemesderfer D. Nagy T. DeGray B. Aronson P.S. J. Biol. Chem. 1999; 274: 17518-17524Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Hybridomas were selected based on ability to immunoprecipitate NHE3. mAb was characterized in detail and was to with mAb 10A3 revealed that a significant pool of renal brush border NHE3 resides in oligomeric complexes with megalin (16Biemesderfer D. Nagy T. DeGray B. Aronson P.S. J. Biol. Chem. 1999; 274: 17518-17524Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In the study we a second that was also generated against affinity-purified NHE3 protein complexes and was to immunoprecipitate NHE3. We evaluated whether 1D11 is to NHE3 or to an associated The of the of an immunoblotting in which rabbit renal microvillus membranes were to to a and with mAb a mAb against NHE3, and a polyclonal antibody to brush border membrane the Na/Pi NaPi-2. mAb 1D11 was to a 110-kDa protein that is from the monomeric of NHE3. We whether mAb 1D11 specifically co-precipitates NHE3. in the of are the of an in which rabbit renal microvillus membrane proteins solubilized in were with mAb The complexes were for immunoblotting and with mAb an anti-NHE3 mAb and the 110-kDa 1D11 antigen and NHE3 were co-precipitated by mAb In mAb 1D11 not co-precipitate NaPi-2, indicating that the the 1D11 antigen and NHE3 is In the studies was performed using solubilized microvillus membrane proteins by for we have previously reported that a significant of NHE3 from membranes for a for indicating that of the transporter in the solubilized resides in (16Biemesderfer D. Nagy T. DeGray B. Aronson P.S. J. Biol. Chem. 1999; 274: 17518-17524Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). of NHE3 and the 1D11 antigen in such in for their from the supernatants of renal microvillus membranes in against this possibility is the of of brush border membrane NaPi-2. to that NHE3 and the 1D11 antigen also co-precipitated from the we performed experiments using the and supernatants of solubilized renal microvillus membranes. These supernatants were with a mAb to NHE3, mAb or a mAb to the microvillar protein The were for immunoblotting and with a polyclonal to NHE3. in mAb 1D11 NHE3 from solubilized microvillus membranes but not mAb 1D11 specifically NHE3, we were to demonstrate that the anti-NHE3 mAb 2B9 co-precipitates the 110-kDa 1D11 antigen in this we previously that NHE3 is with the C-terminal for anti-NHE3 mAb 2B9 is from antibody binding (16Biemesderfer D. Nagy T. DeGray B. Aronson P.S. J. Biol. Chem. 1999; 274: 17518-17524Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). We evaluated whether 1D11 also a pool of NHE3 not by anti-NHE3 mAb To this was performed with mAb in of the NHE3 for binding to this as in To whether was NHE3 that was with the 1D11 antigen and that mAb 2B9 was not to a was performed from the using mAb in an of NHE3 that not by anti-NHE3 mAb 2B9 was co-precipitated by mAb These findings indicate that the C-terminal for binding of mAb 2B9 to NHE3 is NHE3 is with the 110-kDa 1D11 demonstrated that the 110-kDa 1D11 antigen and NHE3 are specifically in the characterization of the 1D11 antigen was to the of its expression in proximal tubule cells. by immunoelectron microscopy in mAb 1D11 the brush border of proximal tubule cells. In contrast to the predominantly coated previously for anti-megalin mAb 10A3 (16Biemesderfer D. Nagy T. DeGray B. Aronson P.S. J. Biol. Chem. 1999; 274: 17518-17524Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), mAb 1D11 the microvillar of the brush of the was also The findings in that the 110-kDa 1D11 antigen is a brush border membrane To isolate the protein against which mAb 1D11 is chromatography was by of a to which mAb 1D11 was The antigen from solubilized microvillus membranes was using a The was to and to a The protein was from the blot and for N-terminal The was A indicated that of of the identified amino acids and dipeptidyl peptidase IV (DPPIV, the possibility that DPPIV may have been a in the 110-kDa region of the blot for N-terminal amino we performed of activity to that the protein isolated on the mAb 1D11 microvillus membranes were with the 1D11 of the solubilized microvillus membrane to the the representing solubilized microvillus membranes with the and the from the were for the presence of the 110-kDa 1D11 antigen by Western and for activity in the 1D11 antigen was from the and was in the These findings with of DPPIV activity from the with in the In activity of brush border membrane was not in the and was not in the from the 1D11 These that the 1D11 antigen is In of previous findings that NHE3 is expressed in different microdomains of the apical membrane of proximal tubule cells and that the NHE3-megalin complex principally resides in a dense membrane compartment most likely representing coated pits (17Biemesderfer D. DeGray B. Aronson P.S. J. Biol. Chem. 2001; 276: 10161-10167Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), it was of to in which microdomain the NHE3-DPPIV complex is For this we performed immunoblotting and experiments using microvillus membrane isolated by P.S. J. Biol. PubMed Scopus Google Scholar), and dense membrane isolated by of renal membranes (17Biemesderfer D. DeGray B. Aronson P.S. J. Biol. Chem. 2001; 276: 10161-10167Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The immunoblotting experiments were performed using of protein from or dense to the expression of DPPIV in these in megalin is highly expressed in dense with villin is predominantly expressed in and NHE3 is these microdomains as previously reported (17Biemesderfer D. DeGray B. Aronson P.S. J. Biol. Chem. 2001; 276: 10161-10167Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). We that DPPIV is in than in dense previous findings that DPPIV is a protein of the microvillar microdomain D. G. Aronson P.S. M.G. Am. J. Physiol. Google Scholar). To the of the NHE3-DPPIV complex in these membrane we performed with mAb 1D11 and the for the presence of NHE3 by Western in NHE3 was the in solubilized microvillus membrane and dense a greater of NHE3 was co-precipitated by mAb 1D11 from with dense vesicles. These contrast with previous findings indicating that the NHE3-megalin complex is most in the dense which likely coated pits (17Biemesderfer D. DeGray B. Aronson P.S. J. Biol. Chem. 2001; 276: 10161-10167Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The findings in that the pools of NHE3 associated with DPPIV and megalin have of To the these pools of NHE3, we performed experiments using mAbs 1D11 and 10A3 in precipitation with mAb 1D11 to of the of NHE3 that co-precipitates with To whether was NHE3 that was not associated with DPPIV but was associated with a was performed from the using anti-megalin mAb in a of NHE3 that not by anti-DPPIV mAb 1D11 was co-precipitated by mAb 10A3, indicating that a pool of NHE3 exists that is with megalin but not with in by anti-megalin mAb 10A3, an of NHE3 was by anti-DPPIV mAb indicating that a pool of NHE3 exists that is with DPPIV but not with we that precipitation of megalin from solubilized microvillus membranes by anti-DPPIV mAb 1D11 was and precipitation of DPPIV by anti-megalin mAb 10A3 was not not Taken together, the findings in and that the pools of NHE3 with megalin and DPPIV are and in different microdomains of the apical membrane of proximal tubule cells. In the study we have that mAb 1D11 to DPPIV co-precipitates NHE3 from solubilized microvillus membranes isolated from rabbit that NHE3 and DPPIV are associated in an oligomeric that anti-DPPIV mAb 1D11 co-precipitates villin from solubilized microvillus membranes the of the NHE3 and also known as is a membrane protein expressed in of and nonepithelial cells D. J. D. Exp. PubMed Scopus Google Scholar). is a highly that N-terminal from with a or J. D. E.J. J. PubMed Scopus Google Scholar). this it is to and and such as and on expressed and R. 1999; PubMed Scopus Google Scholar). In to its DPPIV also as a binding the DPPIV to the enzyme J. T. Y. C. 1993; PubMed Scopus Google Scholar), and DPPIV is in of T. H. C. J. Sci. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). is also evidence for a role for in C. Rev. 1998; PubMed Scopus Google Scholar). The kidney is a of expression of it is one of the brush border membrane proteins J. D. E.J. J. 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The apical brush border of proximal tubule cells is of the and the coated pits M.G. J. Biol. PubMed Scopus Google Scholar). We previously demonstrated that NHE3 is in microvillus membrane and in a population of dense membrane that likely the coated pits (17Biemesderfer D. DeGray B. Aronson P.S. J. Biol. Chem. 2001; 276: 10161-10167Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). we identified a pool of NHE3 that is associated in oligomeric complexes with the scavenger receptor megalin and that NHE3-megalin complexes are principally in the coated microdomain of the renal brush border NHE3 in (17Biemesderfer D. DeGray B. Aronson P.S. J. Biol. Chem. 2001; 276: 10161-10167Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). In the we have that the NHE3-DPPIV complex is in NHE3 is than in coated Serial experiments using mAbs 1D11 and 10A3 have revealed that the pools of NHE3 with DPPIV and megalin are A pool of NHE3 by precipitation by anti-NHE3 mAb which co-precipitates megalin DPPIV its the C-terminal of NHE3 is NHE3 is with of these proteins. in the of NHE3 associated with the pool of NHE3 by mAb 2B9 is in than in the microdomain (17Biemesderfer D. DeGray B. Aronson P.S. J. Biol. Chem. 2001; 276: 10161-10167Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). and previous together, are of NHE3 in the renal brush NHE3-megalin complexes that in the coated NHE3-DPPIV complexes that are in the and NHE3 that is also microvillar but is of association with megalin or We have previously that NHE3, of which is with is (17Biemesderfer D. DeGray B. Aronson P.S. J. Biol. Chem. 2001; 276: 10161-10167Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The of NHE3 and NHE3 to the Na+-H+ exchange activity in renal microvillus membrane to is the of of these of NHE3 to the regulatory proteins (11Weinman E.J. Minkoff C. Shenolikar S. Am. J. Physiol. Renal Physiol. 2000; 279: F393-F399Crossref PubMed Google Scholar, 13Weinman E.J. Steplock D. Donowitz M. Shenolikar S. Biochemistry. 2000; 39: 6123-6129Crossref PubMed Scopus (128) Google Scholar) and (14Yun C.H. Oh S. Zizak M. Steplock D. Tsao S. Tse C.M. Weinman E.J. Donowitz M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3010-3015Crossref PubMed Scopus (403) Google Scholar). To we have been to demonstrate of or from renal microvillus membranes by anti-NHE3 mAb 2B9 or anti-megalin mAb 10A3 the we have used for membrane Biemesderfer and P. S. a fraction of renal brush border NHE3 is (16Biemesderfer D. Nagy T. DeGray B. Aronson P.S. J. Biol. Chem. 1999; 274: 17518-17524Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). and the protein D. M. A. J. Biol. 1997; PubMed Scopus Google Scholar, C.H. G. A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, G. Weinman E.J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), it is that in native kidney predominantly with NHE3 that is and is not for the of In we have demonstrated an unexpected association of Na+-H+ exchanger isoform NHE3 with dipeptidyl peptidase IV in microvillus membranes isolated from proximal tubule cells. of DPPIV NHE3 activity in a proximal tubule Aronson P.S. J. Am. Soc. Nephrol. 2000; Scholar). that DPPIV is a protein with known as a binding and findings raise the possibility that association with DPPIV NHE3 by or its surface expression and/or activity.